WO2002000824A2 - Nouveau polypeptide, proteine humaine 10.56 du gene cancerigene tre, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 10.56 du gene cancerigene tre, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000824A2
WO2002000824A2 PCT/CN2001/000936 CN0100936W WO0200824A2 WO 2002000824 A2 WO2002000824 A2 WO 2002000824A2 CN 0100936 W CN0100936 W CN 0100936W WO 0200824 A2 WO0200824 A2 WO 0200824A2
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polypeptide
polynucleotide
human
oncogene protein
sequence
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PCT/CN2001/000936
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English (en)
French (fr)
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WO2002000824A3 (fr
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU89529/01A priority Critical patent/AU8952901A/en
Publication of WO2002000824A2 publication Critical patent/WO2002000824A2/zh
Publication of WO2002000824A3 publication Critical patent/WO2002000824A3/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human tre oncogene protein 10.56, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Tre is a recombinant gene isolated from NIH3T3 cells transfected with human Ewing's sarcoma DNA. It consists of three major genetic components derived from human chromosomes 5, 18 and 17. Tre-2 is a new transcription unit of the tre oncogene, located near the centromere of human chromosome 17q. The same or polymorphic or tumor-specific model of the transcription unit exists in various cancer cells in the human body However, it was not expressed in normal tissue-derived cells.
  • the protein encoded by the tre oncogene is a highly hydrophilic protein with two charged clusters and therefore has the characteristics of binding to nucleotides (Ta tsuya Nakamura, Jana Hi lova, Oncogene (1992), 7, 733 -741).
  • the human tre oncogene protein 10.56 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there is always a need to identify more participants in this field.
  • These processes of human tre oncogene protein 10.56 protein In particular, the amino acid sequence of this protein is identified. Isolation of the new human tre oncogene protein 10.56 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of a diagnostic and / or therapeutic agent for disease 1 and it is therefore important to isolate its coding DM. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human tre oncogene protein 10.56.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human tre oncogene protein 10.56.
  • Another object of the present invention is to provide a method for producing human t re oncogene protein 10.56.
  • Another object of the present invention is to provide a human tre oncogene protein directed to the polypeptide of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the human tre oncogene protein 10.56 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human tre oncogene protein 10.56.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 904 to 1194 in SEQ ID NO: 1; and (b) a sequence having 1 to 1421 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; a package
  • the method of preparing the polypeptide of the present invention includes culturing the host cell and recovering the expressed product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human TRE oncogene protein 10.56 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or disease susceptibility associated with abnormal expression of the human oncogene protein 10.56 protein, comprising detecting mutations in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human oncogene protein 10.56. .
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • “Insertion” or “addition” refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule. "Replacement” refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Biological activity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • the term “immunologically active” refers to the ability of natural, recombinant, or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” is a molecule that, when combined with the human tre oncogene protein 10.56, causes the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds to the human oncogene protein 10.56.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human tre oncogene protein 10.56 when combined with human tre oncogene protein 10.56.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human tre oncogene protein 10.56.
  • Regular refers to a change in the function of human tre oncogene protein 10.56, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological property, function, or immunity of human tre oncogene protein 10.56. Change of nature.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human tre oncogene protein 10.56 using standard protein purification techniques. Basically pure human tre oncogene protein 10.56 produces a single main band on a non-reducing polyacrylamide gel. The purity of the human tre oncogene protein 10.56 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of perfectly homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences interact with each other specifically or selectively.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences (Higg ins, DG and PM Sharp according to different methods such as Clus ter method). (1988) Gene 73: 237-244) 0 C lus ter method by examining the distances between all pairs of each set of sequence arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the number of residues matching between sequence A and sequence B X 100
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or by a method well known in the art Methods such as Jotun He in determine the percent identity between nucleic acid sequences (He in J., (1990) Me thods in emzumo logy 183: 625-645) 0 "similarity" refers to the corresponding position in the alignment of amino acid sequences Degree of identical or conservative substitutions of amino acid residues.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RM sequence.
  • Antisense strand means
  • Sense strand A complementary nucleic acid strand.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa, F (ab ') 2 and Fv, which can specifically bind to the epitope of the human oncogene protein 10.56.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of matter from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides exist in the same state as in a natural state. Separated from other substances, it is isolated and purified.
  • isolated human tre oncogene protein 10.56 refers to human tre oncogene protein 10.56 which is substantially free of other proteins, lipids, sugars or other substances naturally associated with it. Those skilled in the art can purify human tre oncogene protein 10.56 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human tre oncogene protein 10.56 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a human tre oncogene protein 10.56, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the invention can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the human tre oncogene protein 10.56.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human oncogene protein 10.56 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ )
  • Such a polypeptide sequence in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence)
  • such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1421 bases and its open reading frame 904-1194 encodes 96 amino acids.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or Non-coding chain.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fi co ll, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 nucleotides. Nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human tre oncogene protein 10.56.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human oncogene protein 10.56 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DM sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the separation of cDM sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction. Kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of the transcript of human tre oncogene protein 10.56; (4) ) Detection of protein products expressed by genes through immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of 10.56 gene expression of human tre oncogene protein can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Selected and synthesized by conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. To obtain the full-length CDM sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the human tre oncogene protein 10.56 coding sequence, and a method for producing the polypeptide of the present invention by recombinant technology .
  • a polynucleotide sequence encoding a human tre oncogene protein 10.56 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRM synthesis.
  • promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polytumor enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human tre oncogene protein 10.56 or a polynucleotide containing the same The recombinant vector can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS, or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human t oncogene protein 10.56 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromat
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the inventor's tre oncogene protein 10.56 and human tre oncogene protein.
  • the upper graph is a graph of the expression profile of the human tre oncogene protein 10.56, and the lower graph is the graph of the expression profile of the human tre oncogene protein.
  • 1-bladder mucosa 2- PMA + Ecv304 cell line, 3- LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5- Fibroblas t, growth factor stimulation, 1024NT, 6-scarf to fc growth factor Stimulation, 1013HT, 7-scar into fc without stimulation with growth factors, 1013HC, 8-bladder cancer cell EJ, 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12-liver cancer cell line, 13-fetus Skin, 14-spleen, 15-prostate cancer, 16-jejunum adenocarcinoma, 17 cardia cancer.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human tre oncogene protein 10.56.
  • llkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Example 1 Cloning of human tre oncogene protein 10.56
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Quik mRNA I solat ion Kit product of Qiegene was used to isolate poly (A) m 2 2 poly (A) mRNA from total RNA by reverse transcription to form CDM.
  • the Smart cDM cloning kit purchased from Clontech was used to insert the 00 fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5a.
  • the bacteria formed a cDNA library.
  • Dye terminate cycle reaction ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with a public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones G644E09 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer. After purification of Qiagene's kit, PCR amplification was performed with the following primers:
  • Primerl 5'- TACACCGTTAATTTCCTATGGTTG -3 '(SEQ ID NO: 3)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • P mer2 is the 3′-end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50 leg ol / L KC1, 10mmol / L Tris-Cl, (pH8.5), 1.5ramol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol in a 50 ⁇ 1 reaction volume Primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min 0 ⁇ -actin was also set as a positive control during RT-PCR And template blank is negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-1421 bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human tre oncogene protein 10.56 gene expression:
  • RNA extraction in one step involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) are added. ), Mix and centrifuge. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • Electrophoresis was performed on a 1.2% agarose gel containing 2 g of RNA on 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transferred to nitrocellulose.
  • cc- 32 P dATP with 32 P- DNA probes prepared by Method labeled by random priming. The DNA probe used is shown in Figure 1 PCR amplification of the coding region of the human tre 10.56 oncogene protein Sequence (904bp to 1194b P ).
  • 32P-labeled probe (approximately 2 ⁇ 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred in a solution at 42 ° C. overnight, the solution containing 50% formamide -25mM KH 2 P0 ⁇ (pH7.4) -5 ⁇ SSC-5 x Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1 ° / »SDS at 55 ° C 30min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human tre oncogene protein 10.56
  • Primer 3 5, — CCCCATATGATGAGATTTGGGTGGAGTCACAGC- 3, (Seq ID No: 5)
  • Primer 4 5'-CATGGATCCTCACCGTGTTGGCCAGGCTGGCCT-3 '(Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively, followed by the coding sequences of the 5 ' and 3 'ends of the target gene, respectively.
  • the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28M +) (Novagen product, Cat. No. 69865. 3).
  • the pBS-0644E09 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ , containing 10 pg of pBS-0644E09 plasmid, Primer-3 and Primer-4 l Opmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed into colibacillus DH5 c by the calcium chloride method, and cultured overnight in LB plates containing kanamycin (final concentration 3 () ⁇ ⁇ / ⁇ 1), and then positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0644E09) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the host bacteria BL21 (pET-0644E09) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to the final concentration lmraol / L. Continue incubation for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation, and the layers were layered with an affinity column His s. Bind Quick Cartr idge (product of Novagen) capable of binding to 6 histidines (6His-Tag). 56 ⁇ Purification of the target protein human tre oncogene protein 10.56.
  • a peptide synthesizer (product of PE company) was used to synthesize 10.56 specific polypeptide of the following human tre oncogene protein: NH2- Met— Arg-Phe-Gly-Trp- Ser- Hi s- Ser- Gin- Thr- l ie -Leu-Leu- Gin- Trp-C00 H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with 15 ⁇ g / ml bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose (R) column, and the anti-peptide antibody was separated from the total IgG by affinity chromatography.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this example is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps to hybridize the fixed polynucleotide sample to the filter.
  • the sample-immobilized filter is first prehybridized with a probe-free hybridization buffer to saturate the non-specific binding sites of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps. This embodiment uses higher intensity membrane washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention;
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the spot imprint method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other unknown genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 (probel), which belongs to the first type of probe, is identical to the gene fragment of SEQ ID NO: 1 Source or complementary (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Gene chip or gene micro-matrix is a new technology currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze data to To achieve the purpose of fast, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; searching for and screening new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature, for example, see the literature DeRi si, JL, Lyer, V. & Brown, P. 0.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to prepare DNA on a glass slide to prepare a chip. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Ol igotex mRNA Midi Ki t (purchased from QiaGen).
  • the fluorescent test J Cy3dUTP (5-Araino-propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP ( 5- Amino- propargy 1-2'- deoxyur idine 5'-tr iphate coupled to Cy5 f luorescent dye, purchased from Amersham Phamacia Biotech Company, labeled the specific tissue (or stimulated cell line) mRNA of the body, and prepared it after purification Probe.
  • Cy5dUTP 5- Amino- propargy 1-2'- deoxyur idine 5'-tr
  • the probes from the above two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are bladder mucosa, PMA + E CV 304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar formation fc Growth factor stimulation, 1013HT, scar into fc without growth factor stimulation, 1013HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunum adenocarcinoma, cardia cancer. Draw a chart based on these 17 Cy3 / Cy5 ratios. (figure 1 ) . The figure shows that the human tre oncogene protein 10.56 and the human tre oncogene protein expression profile according to the present invention are very similar.
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • tre oncogene This important tumorigenic gene is called tre oncogene, found in human Ewing's sarcoma cells and can be produced in NIH3T3 cells, but has not been found in other human cells It was found that the tre oncogene is a recombinant gene that can be isolated from NIH3T3 cells transfected with human Ewing's sarcoma DNA. Tre-2 is a new transcription unit of the tre oncogene, the same or more of the transcription units State-of-the-art or tumor-specific models exist in various human cancer cells, but are not expressed in normal tissue-derived cells.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human tre oncogene protein, and both have similar biological functions.
  • the polypeptide of the present invention is involved in regulating or activating a variety of human tumor genes in vivo, and its translocation has a very significant activating effect on Ewing's sarcoma as a transcription unit. Its abnormal expression is usually closely related to the occurrence of a variety of tumors, abnormal inflammation or immune disorder. And produce related diseases, especially Ewing's sarcoma.
  • the abnormal expression of the human tre oncogene protein 10.56 of the present invention will produce various diseases, especially bone tumors, other tumors, inflammations, and immune diseases. These diseases include, but are not limited to:
  • Bone tumors Ewing's sarcoma, osteoid osteoma, osteochondroma, chondroma, osteoblastoma, chondroblastoma, etc.), malignant bone tumors such as giant cell tumor of bone, osteosarcoma, chondrosarcoma, myeloma
  • tumors gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, astrocytoma, ependymoma, glioblastoma, neurofibromas, colon cancer, bladder Cancer, endometrial cancer, colon cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • Abnormal expression of the human tre oncogene protein 10.56 of the present invention will also cause certain hereditary, hematological diseases and the like.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially bone tumors, other tumors, inflammation, immune diseases, certain hereditary, blood Sexually transmitted diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human tre oncogene protein 10.56.
  • Agonists enhance the human oncogene protein 10.56 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human tre oncogene protein 10. 56 can be cultured with labeled human tre oncogene protein 10.56 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human oncogene protein 10.56 include screened antibodies, compounds, receptor deletions, and the like. Antagonist of human oncogene protein 10.56 can bind to human oncogene protein 10.56 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the polypeptide Cannot perform biological functions. .
  • human tre oncogene protein 10.56 When screening compounds as antagonists, human tre oncogene protein 10.56 can be added to the bioanalytical assay, and the compound can be determined by determining the effect of the compound on the interaction between human tre oncogene protein 10.56 and its receptor. Whether it is an antagonist. In the same way as above for screening compounds, Receptor deletions and analogs that act as antagonists. Polypeptide molecules capable of binding to human tre oncogene protein 10.56 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 10.56 molecules of human tre oncogene protein should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human tre oncogene protein 10.56 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human tre oncogene protein 10.56 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Wait.
  • Techniques for preparing monoclonal antibodies to human tre oncogene protein 10.56 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV- Hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies U.S. Pat No. 4946778, can also be used to produce single chain antibodies against human tre oncogene protein 10.56.
  • Anti-human tre oncogene protein 10.56 antibody can be used in immunohistochemistry to detect human tre oncogene protein 10.56 in biopsy specimens.
  • Monoclonal antibodies that bind to human tre oncogene protein 10.56 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human tre oncogene protein 10.56 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of the antibody with a thiol crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human tre oncogene protein 10, 56 positive Cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human tre oncogene protein 10.56. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human tre oncogene protein 10.56.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human tre oncogene protein 10.56.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human tre oncogene protein 10.56 detected in the test can be used to explain the human tre oncogene protein 10.56 Importance in various diseases and diseases for which human tre oncogene protein 10.56 plays a role.
  • the polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding human tre oncogene protein 10.56 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human tre oncogene protein 10.56.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human tre oncogene protein 10.56 to inhibit endogenous human tre oncogene protein 10.56 activity.
  • a mutated human tre oncogene protein 10.56 may be a shortened human tre oncogene protein 10.56 lacking a signaling functional domain. Although it can bind to a downstream substrate, it lacks signaling activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human tre oncogene protein 10.56.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human tre oncogene protein 10.56 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human tre oncogene protein 10.56 can be found in the existing literature (Sambrook, et al.). 0
  • Another recombinant polynucleotide encoding human tre oncogene protein 10.56 can be packaged into lipids. In vivo transfer into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit 10.56 mRNA of human tre oncogene protein are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA and DNA and ribozymes can be obtained by any existing RNA or DM synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human tre oncogene protein 10.56 can be used for the diagnosis of diseases related to human tre oncogene protein 10.56.
  • the polynucleotide encoding human tre oncogene protein 10.56 can be used to detect the expression of human tre oncogene protein 10.56 or the abnormal expression of human tre oncogene protein 10.56 in a disease state.
  • the DNA sequence encoding human tre oncogene protein 10.56 can be used to hybridize biopsy specimens to determine the expression of human tre oncogene protein 10.56.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are publicly available Commercially available techniques and related kits are available.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • 56-transcripts of human tre oncogene protein 10.56 can also be detected using in vitro amplification of human tre oncogene protein 10.56 specific primers for MA-polymerase chain reaction (RT-PCR).
  • Human tre oncogene protein 10.56 mutations can also be used to diagnose human tre oncogene protein 10.56-related diseases.
  • Human tre oncogene protein 10.56 mutations include point mutations, translocations, deletions, recombination, and any other abnormalities compared to the normal wild-type human tre oncogene protein 10.56 DM sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • the PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inherance in Man (available online with Johns Hopkins University Welch Medi Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions. Next, the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease.
  • Comparing diseased and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR.
  • the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptide of the present invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human t oncogene protein 10. 56 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of the human oncogene 10.56 to be administered to the patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician

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Description

一种新的多肽一一人 tre致癌基因蛋白 10. 56和编码这种多肽的多核苷酸 技术领域
本发明属于生物技术领域， 具体地说， 本发明描述了一种新的多肽一一人 tre致癌基因蛋白 10. 56 , 以及编码此多肽的多核苷酸序列。 本发明还涉及此多 核苷酸和多肽的制备方法和应用。 背景技术
及非同寻常的骨骼外尤文氏肉瘤中显示了一种连贯的染色体易位 t (11; 22) (q24; ql2) (Aur ias eta l. , 1983; Turc-Carel et a l. , 1983) , 但在小圆 细胞组织的其他肿瘤中未观察到这一现象。 因而， 迄今为止， t ( l l ; 22 )被认 为是诊断尤文氏肉瘤唯一阳性且可靠的标准 （Sandberg et a l . , 1988 ) 。 临床 观察显示染色体易位导致异常地转录激活基因从而启动或引发恶性状态。 这一 重要的具有致瘤效应的基因被称为 tre 致癌基因， 在人体尤文氏肉瘤细胞中发 现， 并可在 NIH3T3细胞中产生， 但在人体其他细胞中未曾发现。
Tre是一种从转染人尤文氏肉瘤 DNA的 NIH3T3细胞中分离得到的重组基因。 它由三种衍生自人染色体 5 , 18及 17的主要基因成分组成。 Tre- 2是 tre致癌 基因的一种新的转录单位， 位于人染色体 17q 的着丝粒近侧区， 该转录单位相 同的或多态的或肿瘤特异的的模型存在于人体的各种癌细胞中， 但在正常的组 织衍生细胞中没有表达。 t re致癌基因编码的蛋白是一种高度亲水性蛋白， 具有 两个带电簇， 因而具有与核苷酸结合的特性 （ Ta tsuya Nakamura, J ana Hi l lova, Oncogene (1992) , 7, 733-741 ) 。
通过基因芯片的分析发现， 在膀胱粘膜、 PMA+的 Ecv304 细胞株、 LPS+的 Ecv304 细胞株胸腺、 正常成纤维细胞 1024NC, Fibroblas t , 生长因子刺激， 1024NT、疤痕成 fc生长因子刺激， 1013HT、疤痕成 f c未用生长因子刺激， 1013HC、 膀胱癌建株细胞 EJ、 膀胱癌旁、 膀胱癌、 肝癌、 肝癌细胞株、 胎皮、 脾脏、 前 列腺癌、 空肠腺癌、 贲门癌中， 本发明的多肽的表达谱与人 tre 致癌基因蛋白 的表达谱非常近似， 因此二者功能也可能类似。 本发明被命名为人 tre 致癌基 因蛋白 10. 56。
由于如上所述人 tre致癌基因蛋白 10. 56蛋白在调节细胞分裂和胚胎发育等 机体重要功能中起重要作用， 而且相信这些调节过程中涉及大量的蛋白， 因而 本领域中一直需要鉴定更多参与这些过程的人 tre致癌基因蛋白 10. 56蛋白， 特别是鉴定这种蛋白的氨基酸序列。 新人 tre致癌基因蛋白 1 0. 56蛋白编码基 因的分离也为研究确定该蛋白在健康和疾病状态下的作用提供了基础。 这种蛋 白可能构成开发疾 1病诊断和 /或治疗药的基础， 因此分离其编码 DM是非常重 要的。 发明的公开
本发明的一个目的是提供分离的新的多肽一一人 tre致癌基因蛋白 10. 56以 及其片段、 类似物和衍生物。
本发明的另一个目的是提供编码该多肽的多核苷酸。
本发明的另一个目的是提供含有编码人 tre致癌基因蛋白 10. 56的多核苷酸 的重组载体。
本发明的另一个目的是提供含有编码人 tre致癌基因蛋白 10. 56的多核苷酸 的基因工程化宿主细胞。
本发明的另一个目的是提供生产人 t re致癌基因蛋白 1 0. 56的方法。
本发明的另一个目的是提供针对本发明的多肽一一人 tre 致癌基因蛋白
10. 56的抗体。
本发明的另一个目的是提供了针对本发明多肽一一人 t re 致癌基因蛋白 10. 56的模拟化合物、 拮抗剂、 激动剂、 抑制剂。
本发明的另一个目的是提供诊断治疗与人 t re致癌基因蛋白 10. 56异常相关 的疾病的方法。
本发明涉及一种分离的多肽， 该多肽是人源的， 它包含： 具有 SEQ ID No. 2 氨基酸序列的多肽、 或其保守性变体、 生物活性片段或衍生物。 较佳地， 该多 肽是具有 SEQ ID NO: 2氨基酸序列的多肽。
本发明还涉及一种分离的多核苷酸， 它包含选自下组的一种核苷酸序列或 其变体：
(a)编码具有 SEQ ID No. 2氨基酸序列的多肽的多核苷酸；
(b)与多核苷酸（a)互补的多核苷酸；
(c)与（a)或（b)的多核苷酸序列具有至少 70%相同性的多核苷酸。
更佳地， 该多核苷酸的序列是选自下组的一种： （a)具有 SEQ ID NO: 1 中 904- 1194位的序列； 和（b)具有 SEQ ID NO: 1中 1 - 1421位的序列。
本发明另外涉及一种含有本发明多核苷酸的载体， 特别是表达载体； 一种 用该载体遗传工程化的宿主细胞， 包括转化、 转导或转染的宿主细胞； 一种包 括培养所述宿主细胞和回收表达产物的制备本发明多肽的方法。
本发明还涉及一种能与本发明多肽特异性结合的抗体。
本发明还涉及一种筛选的模拟、激活、拮抗或抑制人 t re致癌基因蛋白 10. 56 蛋白活性的化合物的方法， 其包括利用本发明的多肽。 本发明还涉及用该方法 获得的化合物。
本发明还涉及一种体外检测与人 t re致癌基因蛋白 10. 56蛋白异常表达相关 的疾病或疾病易感性的方法，包括检测生物样品中所述多肽或其编码多核苷酸序列 中的突变， 或者检测生物样品中本发明多肽的量或生物活性。
本发明也涉及一种药物组合物， 它含有本发明多肽或其模拟物、 激活剂、 拮 抗剂或抑制剂以及药学上可接受的载体。
本发明还涉及本发明的多肽和 /或多核苷酸在制备用于治疗癌症、发育性疾 病或免疫性疾病或其它由于人 t re致癌基因蛋白 1 0. 56表达异常所引起疾病的 药物的用途。
本发明的其它方面由于本文的技术的公开，对本领域的技术人员而言是显而易 见的。
本说明书和权利要求书中使用的下列术语除非特别说明具有如下的含义： "核酸序列" 是指寡核苷酸、 核苷酸或多核苷酸及其片段或部分， 也可以指 基因组或合成的 DNA或 RNA , 它们可以是单链或双链的， 代表有义链或反义链。 类似地， 术语 "氨基酸序列" 是指寡肽、 肽、 多肽或蛋白质序列及其片段或部 分。 当本发明中的 "氨基酸序列" 涉及一种天然存在的蛋白质分子的氨基酸序 列时， 这种 "多肽" 或 "蛋白质" 不意味着将氨基酸序列限制为与所述蛋白质 分子相关的完整的天然氨基酸。
蛋白质或多核苷酸 "变体" 是指一种具有一个或多个氨基酸或核苷酸改变 的氨基酸序列或编码它的多核苷酸序列。 所述改变可包括氨基酸序列或核苷酸 序列中氨基酸或核苷酸的缺失、 插入或替换。 变体可具有 "保守性" 改变， 其 中替换的氨基酸具有与原氨基酸相类似的结构或化学性质， 如用亮氨酸替换异 亮氨酸。 变体也可具有非保守性改变， 如用色氨酸替换甘氨酸。
"缺失"是指在氨基酸序列或核苷酸序列中一个或多个氨基酸或核苷酸的缺 失。
"插入" 或 "添加" 是指在氨基酸序列或核苷酸序列中的改变导致与天然存在 的分子相比， 一个或多个氨基酸或核苷酸的增加。 "替换 " 是指由不同的氨基酸或 核苷酸替换一个或多个氨基酸或核苷酸。 "生物活性" 是指具有天然分子的结构、 调控或生物化学功能的蛋白质。 类似 地， 术语 "免疫学活性" 是指天然的、 重组的或合成蛋白质及其片段在合适的动物 或细胞中诱导特定免疫反应以及与特异性抗体结合的能力。
"激动剂" 是指当与人 tre致癌基因蛋白 10. 56结合时， 一种可引起该蛋白质 改变从而调节该蛋白质活性的分子。 激动剂可以包括蛋白质、 核酸、 碳水化合 物或任何其它可结合人 tre致癌基因蛋白 10. 56的分子。
"拮抗剂" 或 "抑制物" 是指当与人 tre致癌基因蛋白 10. 56结合时， 一种可 封闭或调节人 t re致癌基因蛋白 10. 56的生物学活性或免疫学活性的分子。 拮抗 剂和抑制物可以包括蛋白质、 核酸、 碳水化合物或任何其它可结合人 tre致癌基 因蛋白 10. 56的分子。
"调节" 是指人 tre致癌基因蛋白 10. 56的功能发生改变， 包括蛋白质活性的 升高或降低、 结合特性的改变及人 tre致癌基因蛋白 10. 56的任何其它生物学性 质、 功能或免疫性质的改变。
"基本上纯"是指基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化人 tre致癌基因蛋白 10. 56。 基 本上纯的人 tre致癌基因蛋白 10. 56在非还原性聚丙烯酰胺凝胶上能产生单一的主 带。 人 tre致癌基因蛋白 10. 56多肽的纯度可用氨基酸序列分析。
"互补的" 或 "互补" 是指在允许的盐浓度和温度条件下通过碱基配对的多 核苷酸天然结合。 例如， 序列 "C- T- G- A" 可与互补的序列 "G-A- C-T" 结合。 两个单链分子之间的互补可以是部分的或全部的。 核酸链之间的互补程度对于 核酸链之间杂交的效率及强度有明显影响。
"同源性" 是指互补的程度， 可以是部分同源或完全同源。 "部分同源" 是 指一种部分互补的序列， 其至少可部分抑制完全互补的序列与靶核酸的杂交。 这种杂交的抑制可通过在严格性程度降低的条件下进行杂交 （Southern印迹或 Northern印迹等） 来检测。 基本上同源的序列或杂交探针可竞争和抑制完全同 源的序列与靶序列在的严格性程度降低的条件下的结合。 这并不意味严格性程 度降低的条件允许非特异性结合， 因为严格性程度降低的条件要求两条序列相 互的结合为特异性或选择性相互作用。
"相同性百分率"是指在两种或多种氨基酸或核酸序列比较中序列相同或相 似的百分率。可用电子方法测定相同性百分率，如通过 MEGALIGN程序（Lasergene sof tware package, DNASTAR, Inc. , Madi son Wi s. ) 。 MEGALIGN程序可根据不 同的方法如 Clus ter法比较两种或多种序列（Higg ins , D. G. 和 P. M. Sharp (1988) Gene 73: 237-244) 0 C lus ter法通过检查所有配对之间的距离将各组序 列排列成簇。 然后将各簇以成对或成组分配。 两个氨基酸序列如序列 A和序列 B 之间的相同性百分率通过下式计算：
序列 A与序列 B之间匹配的残基个数 X 100 序列 A的残基数一序列 A中间隔残基数一序列 B中间隔残基数 也可以通过 C lus ter法或用本领域周知的方法如 Jotun He in测定核酸序列之 间的相同性百分率（He in J. , (1990) Me thods in emzumo logy 183: 625-645) 0 "相似性" 是指氨基酸序列之间排列对比时相应位置氨基酸残基的相同或 保守性取代的程度。 用于保守性取代的氨基酸例如， 带负电荷的氨基酸可包括 天冬氨酸和谷氨酸； 带正电荷的氨基酸可包括赖氨酸和精氨酸； 具有不带电荷 的头部基团有相似亲水性的氨基酸可包括亮氨酸、 异亮氨酸和缬氨酸； 甘氨酸 和丙氨酸； 天冬酰胺和谷氨酰胺； 丝氨酸和苏氨酸； 苯丙氨酸和酪氨酸。
"反义" 是指与特定的 DNA或 RM序列互补的核苷酸序列。 "反义链" 是指与
"有义链" 互补的核酸链。
"衍生物" 是指 HFP或编码其的核酸的化学修饰物。 这种化学修饰物可以是 用烷基、 酰基或氨基替换氢原子。 核酸衍生物可编码保留天然分子的主要生物 学特性的多肽。
"抗体" 是指完整的抗体分子及其片段， 如 Fa、 F (ab') 2及 Fv, 其能特异性 结合人 t re致癌基因蛋白 10. 56的抗原决定簇。
"人源化抗体" 是指非抗原结合区域的氨基酸序列被替换变得与人抗体更 为相似， 但仍保留原始结合活性的抗体。
"分离的" 一词指将物质从它原来的环境 （例如， 若是自然产生的就指其天 然环境） 之中移出。 比如说， 一个自然产生的多核苷酸或多肽存在于活动物中 就是没有被分离出来， 但同样的多核苷酸或多肽同一些或全部在自然系统中与 之共存的物质分开就是分离的。 这样的多核苷酸可能是某一载体的一部分， 也 可能这样的多核苷酸或多肽是某一组合物的一部分。 既然载体或组合物不是它 天然环境的成分， 它们仍然是分离的。
如本发明所用， "分离的"是指物质从其原始环境中分离出来（如果是天然 的物质， 原始环境即是天然环境） 。 如活体细胞内的天然状态下的多聚核苷酸 和多肽是没有分离纯化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在 的其他物质中分开， 则为分离纯化的。
如本文所用， "分离的人 t re致癌基因蛋白 10. 56" 是指人 t re致癌基因蛋 白 10. 56基本上不含天然与其相关的其它蛋白、 脂类、 糖类或其它物质。 本领域 的技术人员能用标准的蛋白质纯化技术纯化人 t re致癌基因蛋白 10. 56。 基本上 纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。 人 t re 致癌基因蛋白 10. 56多肽的纯度能用氨基酸序列分析。
本发明提供了一种新的多肽一一人 tre致癌基因蛋白 10. 56 , 其基本上是由 SEQ ID NO: 2所示的氨基酸序列组成的。 本发明的多肽可以是重组多肽、 天然多肽、 合 成多肽， 优选重组多肽。 本发明的多肽可以是天然纯化的产物， 或是化学合成的产 物， 或使用重组技术从原核或真核宿主 (例如， 细菌、 酵母、 高等植物、 昆虫和哺 乳动物细胞）中产生。 根据重组生产方案所用的宿主， 本发明的多肽可以是糖基化 的， 或可以是非糖基化的。 本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括人 tre致癌基因蛋白 1 0. 56 的片段、 衍生物和类似物。 如本 发明所用， 术语 "片段" 、 "衍生物" 和 "类似物" 是指基本上保持本发明的 人 t re致癌基因蛋白 10. 56相同的生物学功能或活性的多肽。 本发明多肽的片 段、 衍生物或类似物可以是： （I )这样一种， 其中一个或多个氨基酸残基被保 守或非保守氨基酸残基 （优选的是保守氨基酸残基） 取代， 并且取代的氨基酸 可以是也可以不是由遗传密码子编码的； 或者 （ Π ) 这样一种， 其中一个或多 个氨基酸残基上的某个基团被其它基团取代包含取代基； 或者（ Π Ι )这样一种， 其中成熟多肽与另一种化合物 （比如延长多肽半衰期的化合物， 例如聚乙二醇） 融合； 或者 （ IV ) 这样一种， 其中附加的氨基酸序列融合进成熟多肽而形成的 多肽序列 （如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列） 通 过本文的阐述， 这样的片段、 衍生物和类似物被认为在本领域技术人员的知识 范围之内。
本发明提供了分离的核酸（多核苷酸） ， 基本由编码具有 SEQ ID NO: 2 氨 基酸序列的多肽的多核苷酸组成。 本发明的多核苷酸序列包括 SEQ ID NO: 1 的 核苷酸序列。 本发明的多核苷酸是从人胎脑组织的 cDNA文库中发现的。 它包含 的多核苷酸序列全长为 1421个碱基，其开放读框 904-1194编码了 96个氨基酸。 根据基因芯片表达谱比较发现， 此多肽与人 t re致癌基因蛋白有相似的表达谱， 可推断出该人 t re致癌基因蛋白 1 0. 56具有人 t re致癌基因蛋白相似的功能。
本发明的多核苷酸可以是 DNA形式或是 RNA形式。 DNA形式包括 cDNA、 基因 组 DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或 非编码链。 编码成熟多肽的编码区序列可以与 SEQ ID N0: 1 所示的编码区序列 相同或者是简并的变异体。 如本发明所用， "简并的变异体" 在本发明中是指 编码具有 SEQ ID NO: 2 的蛋白质或多肽， 但与 SEQ ID NO: 1所示的编码区序列 有差别的核酸序列。
编码 SEQ ID NO: 2的成熟多肽的多核苷酸包括： 只有成熟多肽的编码序列； 成熟多肽的编码序列和各种附加编码序列； 成熟多肽的编码序列 （和任选的附 加编码序列） 以及非编码序列。
术语 "编码多肽的多核苷酸" 是指包括编码此多肽的多核苷酸和包括附加 编码和 /或非编码序列的多核苷酸。
本发明还涉及上述描述多核苷酸的变异体， 其编码与本发明有相同的氨基 酸序列的多肽或多肽的片断、 类似物和衍生物。 此多核苷酸的变异体可以是天 然发生的等位变异体或非天然发生的变异体。 这些核苷酸变异体包括取代变异 体、 缺失变异体和插入变异体。 如本领域所知的， 等位变异体是一个多核苷酸 的替换形式， 它可能是一个或多个核苷酸的取代、 缺失或插入， 但不会从实质 上改变其编码的多肽的功能。
本发明还涉及与以上所描述的序列杂交的多核苷酸 （两个序列之间具有至 少 50%, 优选具有 70%的相同性） 。 本发明特别涉及在严格条件下与本发明所述 多核苷酸可杂交的多核苷酸。 在本发明中， "严格条件" 是指： （1)在较低离子 强度和较高温度下的杂交和洗脱， 如 0. 2xSSC, 0. 1%SDS, 60 °C ;或（2)杂交时加用 变性剂， 如 50% (v/v)甲酰胺， 0. 1%小牛血清 / 0. l%Fi co l l , 42 °C等; 或（3)仅在 两条序列之间的相同性至少在 95%以上，更好是 97%以上时才发生杂交。并且， 可 杂交的多核苷酸编码的多肽与 SEQ ID NO: 2所示的成熟多肽有相同的生物学功 能和活性。
本发明还涉及与以上所描述的序列杂交的核酸片段。 如本发明所用， "核酸 片段"的长度至少含 1 0个核苷酸，较好是至少 20- 30个核苷酸，更好是至少 50 - 60 个核苷酸，最好是至少 100个核苷酸以上。核酸片段也可用于核酸的扩增技术（如 PCR)以确定和 /或分离编码人 t re致癌基因蛋白 1 0. 56的多核苷酸。
本发明中的多肽和多核苷酸优选以分离的形式提供， 更佳地被纯化至均质。 本发明的编码人 t re致癌基因蛋白 1 0. 56的特异的多核苷酸序列能用多种方 法获得。 例如， 用本领域熟知的杂交技术分离多核苷酸。 这些技术包括但不局限 于： 1)用探针与基因组或 cDNA文库杂交以检出同源的多核苷酸序列， 和 2)表达 文库的抗体筛选以检出具有共同结构特征的克隆的多核苷酸片段。 本发明的 DM片段序列也能用下列方法获得： 1)从基因组 DNA分离双链 DM 序列； 2)化学合成 DNA序列以获得所述多肽的双链 DNA。
上述提到的方法中， 分离基因组 DM最不常用。 DNA序列的直接化学合成是 经常选用的方法。 更经常选用的方法是 cDM序列的分离。 分离感兴趣的 cDNA的 标准方法是从高表达该基因的供体细胞分离 mRNA并进行逆转录， 形成质粒或噬 菌体 cDNA文库。 提取 mRNA的方法巳有多种成熟的技术， 试剂盒也可从商业途径 获得（Qiagene)。而构建 cDNA文库也是通常的方法（Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989)。 还可得到商业供应的 cDNA文库， 如 Clontech公司的不同 cDNA文库。 当 结合使用聚合酶反应技术时， 即使极少的表达产物也能克隆。
可用常规方法从这些 cDNA文库中筛选本发明的基因。这些方法包括（但不限 于）： （l)DNA-DNA或 DNA- RNA杂交； （2)标志基因功能的出现或丧失； （3)测定人 tre 致癌基因蛋白 10.56 的转录本的水平； （4)通过免疫学技术或测定生物学活 性， 来检测基因表达的蛋白产物。 上述方法可单用， 也可多种方法联合应用。
在第（1)种方法中， 杂交所用的探针是与本发明的多核苷酸的任何一部分同 源， 其长度至少 10个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷 酸， 最好是至少 100个核苷酸。 此外， 探针的长度通常在 2000个核苷酸之内， 较佳的为 1000个核苷酸之内。 此处所用的探针通常是在本发明的基因序列信息 的基础上化学合成的 DM序列。 本发明的基因本身或者片段当然可以用作探针。 DNA探针的标记可用放射性同位素， 荧光素或酶（如碱性磷酸酶）等。
在第（4)种方法中， 检测人 tre致癌基因蛋白 10.56基因表达的蛋白产物可 用免疫学技术如 Western印迹法， 放射免疫沉淀法， 酶联免疫吸附法（ELISA)等。
应 用 PCR 技术 扩增 DNA/RNA 的 方 法 （Saiki, et al. Science 1985; 230: 1350- 1354)被优选用于获得本发明的基因。 特别是很难从文库中得到 全长的 cDNA 时， 可优选使用 RACE法（RACE - cDNA末端快速扩增法）， 用于 PCR 的引物可根据本文所公开的本发明的多核苷酸序列信息适当地选择，并可用常规 方法合成。 可用常规方法如通过凝胶电泳分离和纯化扩增的 DNA/RNA片段。
如上所述得到的本发明的基因，或者各种 DNA片段等的多核苷酸序列可用常 规方法如双脱氧链终止法（Sanger et al. PNAS, 1977, 74: 5463-5467)测定。 这类多核苷酸序列测定也可用商业测序试剂盒等。为了获得全长的 cDM序列， 测 序需反复进行。 有时需要测定多个克隆的 cDNA序列， 才能拼接成全长的 cDNA序 列。 本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或直接用 人 tre致癌基因蛋白 10.56编码序列经基因工程产生的宿主细胞，以及经重组技 术产生本发明所述多肽的方法。
本发明中，编码人 tre致癌基因蛋白 10.56的多核苷酸序列可插入到载体中， 以构成含有本发明所述多核苷酸的重组载体。 术语 "载体" 指本领域熟知的细菌 质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒如腺病毒、 逆转录 病毒或其它载体。 在本发明中适用的载体包括但不限于： 在细菌中表达的基于 T7 启动子的表达载体（Rosenberg, et al. Gene, 1987, 56: 125); 在哺乳动物 细胞中表达的 pMSXND表达载体（Lee and Nathans, J Bio Chem. 263: 3521, 1988) 和在昆虫细胞中表达的来源于杆状病毒的载体。 总之， 只要能在宿主体内复制和 稳定， 任何质粒和载体都可以用于构建重组表达载体。 表达载体的一个重要特征 是通常含有复制起始点、 启动子、 标记基因和翻译调控元件。
本领域的技术人员熟知的方法能用于构建含编码人 tre致癌基因蛋白 10.56 的 DNA序列和合适的转录 /翻译调控元件的表达载体。这些方法包括体外重组 DNA 技术、 DNA合成技术、 体内重组技术等（Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989)。 所述的 DM序列可有效连接到表达载体中的适当启动子上， 以指导 mRM合成。 这些启动子的代表性例子有： 大肠杆菌的 lac或 trp启动子； λ噬菌体的 PL启 动子； 真核启动子包括 CMV立即早期启动子、 HSV胸苷激酶启动子、 早期和晚期 SV40启动子、 反转录病毒的 LTRs和其它一些已知的可控制基因在原核细胞或真 核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点 和转录终止子等。在载体中插入增强子序列将会使其在高等真核细胞中的转录得 到增强。 增强子是 DNA表达的顺式作用因子， 通常大约有 10到 300个碱基对， 作用于启动子以增强基因的转录。 可举的例子包括在复制起始点晚期一侧的 100 到 270个碱基对的 SV40增强子、 在复制起始点晚期一侧的多瘤增强子以及腺病 毒增强子等。
此外，表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转 化的宿主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗性以 及绿色荧光蛋白（GFP) , 或用于大肠杆菌的四环素或氨苄青霉素抗性等。
本领域一般技术人员都清楚如何选择适当的载体 /转录调控元件 （如启动 子、 增强子等） 和选择性标记基因。
本发明中，编码人 tre致癌基因蛋白 10.56的多核苷酸或含有该多核苷酸的 重组载体可转化或转导入宿主细胞，以构成含有该多核苷酸或重组载体的基因工 程化宿主细胞。 术语 "宿主细胞"指原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 细菌细胞如鼠伤寒沙门氏菌； 真菌细胞如酵母； 植物细胞； 昆虫细胞 如果蝇 S2或 Sf 9 ; 动物细胞如 CH0、 COS或 Bowes黑素瘤细胞等。
用本发明所述的 DNA序列或含有所述 DNA序列的重组载体转化宿主细胞可用 本领域技术人员熟知的常规技术进行。 当宿主为原核生物如大肠杆菌时， 能吸收 DM的感受态细胞可在指数生长期后收获， 用 CaCl2法处理， 所用的步骤在本领 域众所周知。 可供选择的是用 MgCl2。 如果需要， 转化也可用电穿孔的方法进行。 当宿主是真核生物， 可选用如下的 DM转染方法： 磷酸钙共沉淀法， 或者常规机 械方法如显微注射、 电穿孔、 脂质体包装等。
通过常规的重组 DM技术，利用本发明的多核苷酸序列可用来表达或生产重 组的人 t re致癌基因蛋白 1 0. 56 (Sc i ence , 1984 ; 224 : 1431)。 一般来说有以下 步骤：
(1) .用本发明的编码人 人 t re致癌基因蛋白 1 0. 56的多核苷酸 (或变异体）， 或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2) .在合适的培养基中培养宿主细胞；
(3) .从培养基或细胞中分离、 纯化蛋白质。
在步骤（2 )中， 根据所用的宿主细胞， 培养中所用的培养基可选自各种常 规培养基。 在适于宿主细胞生长的条件下进行培养。 当宿主细胞生长到适当的细 胞密度后， 用合适的方法（如温度转换或化学诱导）诱导选择的启动子， 将细胞再 培养一段时间。
在步骤 （3 ) 中， 重组多肽可包被于细胞内、 或在细胞膜上表达、 或分泌到 细胞外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离 和纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法包括但并不 限于： 常规的复性处理、 蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超声波 处理、 超离心、 分子筛层析 (凝胶过滤）、 吸附层析、 离子交换层析、 高效液相层 析（HPLC)和其它各种液相层析技术及这些方法的结合。 附图的简要说明
下列附图用于说明本发明的具体实施方案， 而不用于限定由杈利要求书所 界定的本发明范围。 图 1是本发明人 tre致癌基因蛋白 10. 56和人 tre致癌基因蛋白的基因芯片表达 谱比较图。 上图是人 tre致癌基因蛋白 10. 56的表达谱折方图， 下图是人 tre致癌基 因蛋白的表达谱折方图。 其中， 1-膀胱粘膜、 2- PMA+的 Ecv304细胞株、 3- LPS+的 Ecv304细胞株胸腺、 4-正常成纤维细胞 1024NC、 5- Fibroblas t， 生长因子刺激， 1024NT、6-疤痕成 fc生长因子刺激，1013HT、7-疤痕成 fc未用生长因子刺激， 1013HC、 8 -膀胱癌建株细胞 EJ、 9-膀胱癌旁、 10-膀胱癌、 11-肝癌、 12-肝癌细胞株、 13- 胎皮、 14-脾脏、 15-前列腺癌、 16-空肠腺癌、 17贲门癌。
图 2为分离的人 tre致癌基因蛋白 10. 56的聚丙烯酰胺凝胶电泳图（SDS-PAGE )。 llkDa为蛋白质的分子量。 箭头所指为分离出的蛋白条带。 实现本发明的最佳方式
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件如 Sambrook等人， 分子克隆： 实验室手册（New York: Cold Spr ing Harbor Labora tory Pres s, 1989)中所述的条件， 或按照制造厂商所建 议的条件。
实施例 1 : 人 tre致癌基因蛋白 10. 56的克隆
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA。 用 Quik mRNA I solat ion Ki t ( Qiegene 公司产品） 从总 RNA中分离 poly (A) m氣 2ug poly (A) mRNA经逆转录 形成 cDM。用 Smart cDM克隆试剂盒（购自 Clontech )将00 片段定向插入到 pBSK (+) 载体 (Clontech公司产品）的多克隆位点上， 转化 DH5 a , 细菌形成 cDNA文库。 用 Dye terminate cycle react ion sequencing ki t (Perkin-Elmer公司产品) 和 ABI 377 自动测序仪 (Perkin-Elmer公司）测定所有克隆的 5'和 3'末端的序列。 将测定的 cDNA 序列与巳有的公共 DNA序列数据库 （Genebank )进行比较， 结果发现其中一个克隆 G644E09的 cDNA序列为新的 DNA。 通过合成一系列引物对该克隆所含的插入 cDNA片段 进行双向测定。 结果表明， 0644E09克隆所含的全长 cDNA为 1421bp (如 Seq ID NO: 1 所示） ， 从第 904bp至 1194bp有一个 290bp的开放阅读框架 （ 0RF ) , 编码一个新的 蛋白质 （如 Seq ID NO: 2所示） 。 我们将此克隆命名为 pBS - 0644E09 , 编码的蛋白质 命名为人 tre致癌基因蛋白 10. 56。 实施例 2: 用 RT-PCR方法克隆编码人 tre致癌基因蛋白 10. 56的基因
用胎脑细胞总 RNA为模板， 以 ol igo-dT为引物进行逆转录反应合成 cDNA，用 Qiagene的试剂盒纯化后，用下列引物进行 PCR扩增：
Primerl: 5'- TACACCGTTAATTTCCTATGGTTG -3' (SEQ ID NO: 3)
Primer 2: 5，- TTTTTTTCTTTCTTTCTTTTTTTT -3' (SEQ ID NO: 4)
Primerl为位于 SEQ ID NO: 1的 5'端的第 lbp开始的正向序列；
P mer2为 SEQ ID NO: 1的中的 3'端反向序列。
扩增反应的条件： 在 50 μ 1的反应体积中含有 50腿 ol/L KC1, 10mmol/L Tris-Cl, (pH8.5) , 1.5ramol/L MgCl2, 200 μ mol/L dNTP, lOpmol引物， 1U的 Taq DNA 聚合酶（Clontech公司产品）。 在 PE9600型 DNA热循环仪（Perkin- Elmer公司）上按下 列条件反应 25个周期： 94°C 30sec; 55°C 30sec; 72°C 2min0 在 RT- PCR时同时设 β -actin为阳性对照和模板空白为阴性对照。 扩增产物用 QIAGEN公司的试剂盒纯化， 用 TA克隆试剂盒连接到 pCR载体上（Invitrogen公司产品） 。 DNA序列分析结果表明 PCR产物的 DNA序列与 SEQ ID NO: 1所示的 1- 1421bp完全相同。 实施例 3: Northern 印迹法分析人 tre致癌基因蛋白 10.56基因的表达：
用一步法提取总 RNA [Anal. Biochem 1987, 162,156-159]。 该法包括酸性硫氰 酸胍苯酚-氯仿抽提。 即用 4M异硫氰酸胍- 25mM柠檬酸纳， 0.2M乙酸钠 （ pH4.0 ) 对 组织进行匀浆， 加入 1倍体积的苯酚和 1/5体积的氯仿-异戊醇 （49: 1) , 混合后离 心。 吸出水相层， 加入异丙醇 （0.8体积）并将混合物离心得到 RNA沉淀。 将得到的 RNA沉淀用 70%乙醇洗涤， 干燥并溶于水中。 用 2(^gRNA, 在含 20mM 3- ( N-吗啉代） 丙磺酸 (pH7.0) -5mM乙酸钠 - ImM EDTA- 2.2M甲醛的 1.2%琼脂糖凝胶上进行电泳。 然后转移至硝酸纤维素膜上。用 cc- 32P dATP通过随机引物法制备 32P-标记的 DNA探针。 所用的 DNA探针为图 1所示的 PCR扩增的人 tre致癌基因蛋白 10.56编码区序列（904bp 至 1194bP)。 将 32P-标记的探针 （约 2xl06cpm/ml) 与转移了 RNA的硝酸纤维素膜在 一溶液中于 42°C杂交过夜， 该溶液包含 50%甲酰胺 -25mM KH2P0< ( pH7.4 ) -5 χ SSC-5 xDenhardt's溶液和 200 g/ml鲑精 DNA。 杂交之后， 将滤膜在 1 x SSC-0.1°/»SDS中于 55°C洗 30min。 然后， 用 Phosphor Imager进行分析和定量。 实施例 4: 重组人 tre致癌基因蛋白 10.56的体外表达、 分离和纯化
根据 SEQ ID NO: 1和图 1所示的编码区序列， 设计出一对特异性扩增引物， 序列 如下：
Primer 3: 5，— CCCCATATGATGAGATTTGGGTGGAGTCACAGC- 3， ( Seq ID No: 5 ) Primer4: 5'-CATGGATCCTCACCGTGTTGGCCAGGCTGGCCT-3' (Seq ID No: 6 ) 此两段引物的 5'端分别含有 Ndel和 BamHI酶切位点，其后分别为目的基因 5'端和 3'端的编码序列， Ndel和 BamHI酶切位点相应于表达载体质粒 pET- 28M+) (Novagen 公司产品， Cat. No. 69865. 3)上的选择性内切酶位点。 以含有全长目的基因的 pBS- 0644E09质粒为模板， 进行 PCR反应。 PCR反应条件为： 总体积 50 μ ΐ中含 pBS - 0644Ε09质粒 10pg、 引物 Primer- 3和 Primer-4分别为 l Opmol、 Advantage polymerase Mix ( Clontech公司产品） 1 μ 1。 循环参数： 94°C 20s, 60°C 30s, 68°C 2 min,共 25个循环。 用 Ndel和 BamHI分别对扩增产物和质粒 pET- 28 (+)进行双酶切，分 别回收大片段，并用 T4连接酶连接。 连接产物转化用氯化钙法大肠杆细菌 DH5 c ,在 含卡那霉素 （终浓度 3() μ §/ιη1 ) 的 LB平板培养过夜后， 用菌落 PCR方法筛选阳性克 隆， 并进行测序。 挑选序列正确的阳性克隆（pET- 0644E09 ) 用氯化钙法将重组质 粒转化大肠杆菌 BL21 (DE3) plySs (Novagen公司产品）。 在含卡那霉素 （终浓度 30 μ g/ml ) 的 LB液体培养基中， 宿主菌 BL21 ( pET-0644E09 )在 37°C培养至对数生长期， 加入 IPTG至终浓度 lmraol/L， 继续培养 5小时。 离心收集菌体， 经超声波破菌，离心 收集上清， 用能与 6个组氨酸 ( 6Hi s-Tag ) 结合的亲和层析柱 Hi s. Bind Quick Cartr idge ( Novagen公司产品）进行层析， 得到了纯化的目的蛋白人 tre致癌基因 蛋白 10. 56。 经 SDS-PAGE电泳， 在 llkDa处得到一单一的条带 （图 2 ) 。 将该条带转 移至 PVDF膜上用 Edams水解法进行 N-端氨基酸序列分析， 结果 N-端 15个氨基酸与 SEQ ID NO: 2所示的 N-端 15个氨基酸残基完全相同。 实施例 5 抗人 tre致癌基因蛋白 10. 56抗体的产生
用多肽合成仪（PE公司产品）合成下述人 tre致癌基因蛋白 10. 56特异性的多肽： NH2- Met— Arg-Phe-Gly-Trp- Ser- Hi s- Ser- Gin- Thr- l ie- Leu-Leu- Gin- Trp-C00 H (SEQ ID NO: 7)。 将该多肽分别与血蓝蛋白和牛血清白蛋白耦合形成复合， 方法 参见： Avrameas, et al. Immunochemis try, 1969; 6: 43。 用 4mg上述血蓝蛋白多肽复 合物加上完全弗氏佐剂免疫家兔， 15天后再用血蓝蛋白多肽复合物加不完全弗氏佐 剂加强免疫一次。 采用经 15 μ g/ml牛血清白蛋白多肽复合物包被的滴定板做 ELISA 测定兔血清中抗体的滴度。 用蛋白 A-Sepharose从抗体阳性的家兔血清中分离总 IgG。 将多肽结合于溴化氰活化的 Sepharose^柱上， 用亲和层析法从总 IgG中分离 抗多肽抗体。 免疫沉淀法证明纯化的抗体可特异性地与人 tre致癌基因蛋白 10. 56结 合。 实施例 6: 本发明的多核苷酸片段用作杂交探针的应用 从本发明的多核苷酸中挑选出合适的寡核苷酸片段用作杂交探针有多方面的 用途， 如用该探针可与不同来源的正常组织或病理组织的基因组或 cDNA文库杂交 以鉴定其是否含有本发明的多核苷酸序列和检出同源的多核苷酸序列，进一步还可 用该探针检测本发明的多核苷酸序列或其同源的多核苷酸序列在正常组织或病理 组织细胞中的表达是否异常。
本实施例的目的是从本发明的多核苷酸 SEQ ID NO: 1 中挑选出合适的寡核苷 酸片段用作杂交探针，并用滤膜杂交方法鉴定一些组织中是否含有本发明的多核苷 酸序列或其同源的多核苷酸序列。 滤膜杂交方法包括斑点印迹法、 Southern 印迹 法、 Northern 印迹法和复印方法等， 它们都是将待测的多核苷酸样品固定在滤膜 上后使用基本相同的步骤杂交。这些相同的步骤是. · 固定了样品的滤膜首先用不含 探针的杂交缓冲液进行预杂交，以使滤膜上样品的非特异性的结合部位被载体和合 成的多聚物所饱和。然后预杂交液被含有标记探针的杂交缓冲液替换， 并保温使探 针与靶核酸杂交。 杂交步骤之后， 未杂交上的探针被一系列洗膜步骤除掉。 本实施 例利用较高强度的洗膜条件（如较低盐浓度和较高的温度）， 以使杂交背景降低且 只保留特异性强的信号。本实施例选用的探针包括两类： 第一类探针是完全与本发 明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段； 第二类探针是部分与本 发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段。 本实施例选用斑点印 迹法将样品固定在滤膜上，在较高强度的的洗膜条件下， 第一类探针与样品的杂交 特异性最强而得以保留。
一、 探针的选用
从本发明的多核苷酸 SEQ ID NO: 1中选择寡核苷酸片段用作杂交探针， 应遵 循以下原则和需要考虑的几个方面：
1 , 探针大小优选范围为 18- 50个核苷酸；
2 , GC含量为 30%- 70%, 超过则非特异性杂交增加;
3, 探针内部应无互补区域；
4, 符合以上条件的可作为初选探针 , 然后进一步作计算机序列分析， 包括将该初 选探针分别与其来源序列区域 （即 SEQ ID NO: 1 )和其它巳知的基因组序列及 其互补区进行同源性比较， 若与非靶分子区域的同源性大于 85%或者有超过 15 个连续碱基完全相同， 则该初选探针一般就不应该使用；
5, 初选探针是否最终选定为有实际应用价值的探针还应进一步由实验确定。
完成以上各方面的分析后挑选并合成以下二个探针：
探针 1 ( probel ), 属于第一类探针， 与 SEQ ID NO: 1的基因片段完全同 源或互补（41Nt ):
5-TGAGATTTGGGTGGAGTCACAGCCAAACCATATTACTTCAA-3' ( SEQ ID NO: 8 )
探针 2 (probe2)， 属于第二类探针， 相当于 SEQ ID NO: 1的基因片段或 其互补片段的替换突变序列 （41Nt ):
5-TGAGATTTGGGTGGAGTCACCGCCAAACCATATTACTTCAA-3' (SEQ ID NO: 9 ) 与以下具体实验步骤有关的其它未列出的常用试剂及其配制方法请参考文献： DNA PROBES G. H. Keller; M. M. Manak; Stockton Press, 1989 (USA)以及更常用的分 子克隆实验手册书籍如《分子克隆实验指南》（1998年第二版） [美]萨姆布鲁克等 著， 科学出版社。
样品制备：
1, 从新鲜或冰冻组织中提取 DM
步骤： 1 ) 将新鲜或新鲜解冻的正常肝组织放入浸在冰上并盛有磷酸盐缓冲液 (PBS) 的平皿中。 用剪刀或手术刀将组织切成小块。 搡作中应保持组织湿润。 2) 以 lOOOg离心切碎组织 10分钟。 3)用冷匀浆缓冲液 ( 0.25mol/L蔗糖； 25醒 ol/L Tris-HCl,pH7.5; 25隱 ol/LnaCl; 25瞧 ol/L MgCl2 ) 悬浮沉淀 （大约 10ml/g )。 4 ) 在 4°C用电动匀浆器以全速匀浆组织悬液， 直至组织被完全破碎。 5) lOOOg 离心 10分钟。 6)用重悬细胞沉淀（每 O. lg最初组织样品加卜 5ml ), 再以 lOOOg离心 10分钟。 7 ) 用裂解缓冲液重悬沉淀（每 O. lg最初组织样品加 lml ), 然后接以下 的苯酚抽提法。
2, DM的苯酚抽提法
步骤： 1 )用 l-10ml冷 PBS洗细胞， lOOOg离心 10分钟。 2 )用冷细胞裂解液 重悬浮沉淀的细胞（l x 108细胞 /ml )最少应用 lOOul裂解缓冲液。 3)加 SDS至终 浓度为 1°/。, 如果在重悬细胞之前将 SDS直接加入到细胞沉淀中， 细胞可能会形成 大的团块而难以破碎， 并降低的总产率。 这一点在抽提 >107细胞时特别严重。 4) 加蛋白酶 K至终浓度 200ug/ral。 5) 50°C保温反应 1小时或在 37°C轻轻振摇过夜。 6)用等体积苯酚： 氯仿： 异戊醇 U5: 24： 1 )抽提， 在小离心机管中离心 10分 钟。 两相应清楚分离， 否则重新进行离心。 7 )将水相转移至新管。 8)用等体积氯 仿： 异戊醇 （24: 1 )抽提， 离心 10分钟。 9)将含 DNA的水相转移至新管。 然后 进行 DNA的纯化和乙醇沉淀。
3, DNA的纯化和乙醇沉淀
步骤： 1 )将 1/10体积 2mol/L醋酸钠和 2倍体积冷 100°/。乙醇加到 DNA溶液中， 混匀。 在- 20°C放置 1小时或至过夜。 2 ) 离心 10分钟。 3)小心吸出或倒出乙醇。 4)用 70%冷乙醇 500ul洗涤沉淀， 离心 5分钟。 5)小心吸出或倒出乙醇。 用 500ul 冷乙醇洗涤沉淀， 离心 5分钟。 6)小心吸出或倒出乙醇， 然后在吸水纸上倒置使 残余乙醇流尽。 空气干燥 10- 15分钟， 以使表面乙醇挥发。 注意不要使沉淀完全干 燥， 否则较难重新溶解。 7)以小体积 TE或水重悬 DNA沉淀。 低速涡旋振荡或用滴 管吹吸， 同时逐渐增加 TE, 混合至 DNA充分溶解， 每 1-5 106细胞所提取的大约 加 lul。
以下第 8-13步驟仅用于必须除去污染时， 否则可直接进行第 14步骤。
8)将 RNA酶 A加到 DNA溶液中， 终浓度为 100ug/ml, 37°C保温 30分钟。 9 )加入 SDS和蛋白酶 K， 终浓度分别为 0.5%和 100ug/ml。 37°C保温 30分钟。 10)用等体 积的苯酚： 氯仿： 异戊醇（ 25: 24： 1)抽提反应液， 离心 10分钟。 11)小心移出 水相， 用等体积的氯仿： 异戊醇（24: 1)重新抽提， 离心 10分钟。 12)小心移出 水相， 加 1八 0体积 2mol/L醋酸钠和 2.5体积冷乙醇， 混匀置 - 20°C 1小时。 13) 用 70%乙醇及 100%乙醇洗涤沉淀， 空气干燥， 重悬核酸， 过程同第 3-6步骤。 14) 测定 A26。和 A2S。以检测 DM的纯度及产率。 15 )分装后存放于 -20°C。
样膜的制备：
1)取 4x2张适当大小的硝酸纤维素膜（NC膜）， 用铅笔在其上轻轻标出点样位 置及样号， 每一探针需两张 NC膜， 以便在后面的实验步骤中分别用高强度条件和 强度条件洗膜 。
2) 吸取及对照各 15微升， 点于样膜上， 在室温中晾干。
3 ) 置于浸润有 0. Imol/LNaOH, 1.5mol/LNaCl的滤纸上 5分钟 （两次）， 晾干置 于浸润有 0.5mol/L Tris-HCl ( pH7.0 )， 3mol/LNaCl的滤纸上 5分钟 (两次）， 晾 干。
4) 夹于干净滤纸中， 以铝箔包好， 60-80°C真空干燥 2小时。
探针的标记
1 ) 3μ lProbe ( 0.10D/10 μ 1 )，加入 2 μ I inase缓冲液， 8-10 uCi γ- 32P- dATP+2U Kinase, 以补加至终体积 20 μ 1。
2) 37 °C 保温 2小时。
3)加 1/5体积的溴酚蓝指示剂 ( BPB )0
4) it Sephadex G- 50柱。
5 ) 至有 32P- Probe洗出前开始收集第一峰 (可用 Moni tor监测）。
6) 5滴 /管， 收集 10- 15管。
7)用液体闪烁仪监测同位素量 8 )合并第一峰的收集液后即为所需制备的 32P- Probe (第二峰为游离 γ- 32P- dATP )。 预杂交
将样膜置于塑料袋中，加入 3- l Omg预杂交液（ l OxDenhardf s ; 6xSSC, 0. lmg/ml CT DM (小牛胸腺 DNA )。）， 封好袋口后， 68°C水洛摇 2小时。
杂交
将塑料袋剪去一角， 加入制备好的探针， 封好袋口后， 42°C水浴摇过夜。 洗膜：
高强度洗膜：
1 )取出已杂交好的样膜。
2 ) 2xSSC, 0. l°/oSDS中， 40°C洗 15分钟 （ 2次）。
3 ) 0. lxSSC, 0. 1%SDS中， 40°C洗 15分钟 ( 2次）。
4 ) 0. lxSSC, 0. 1%SDS中， 55°C洗 30分钟 （ 2次）， 室温晾干。 低强度洗膜：
1 )取出已杂交好的样膜。
2 ) 2xSSC, 0. 1%SDS中， 37°C洗 15分钟 （ 2次）。
3 ) 0. lxSSC, 0. 1%SDS中， 37°C洗 15分钟 ( 2次）。
4 ) 0. lxSSC, 0. 1 SDS中， 40°C洗 15分钟 （ 2次）， 室温晾干。 X-光自显影：
-70 , X-光自显影 （压片时间根据杂交斑放射性强弱而定）。 釆用' T¼强度洗膜条件所进行的杂交实验，以上两个探针杂交斑放射性强弱没有 明显区别； 而采用高强度洗膜条件所进行的杂交实验，探针 1的杂交斑放射性强度 明显强于另一个探针杂交斑的放射性强度。因而可用探针 1定性和定量地分析本发 明的多核苷酸在不同组织中的存在和差异表达。
实施例 7 DNA Microarray
基因芯片或基因微矩阵（DM Mi croarray )是目前许多国家实验室和大制药公 司都在着手研制和开发的新技术， 它是指将大量的靶基因片段有序地、高密度地排 列在玻璃、硅等载体上， 然后用荧光检测和计算机软件进行数据的比较和分析， 以 达到快速、 高效、 高通量地分析生物信息的目的。 本发明的多核苷酸可作为靶 DNA 用于基因芯片技术用于高通量研究新基因功能；寻找和筛选组织特异性新基因特别 是肿瘤等疾病相关新基因； 疾病的诊断， 如遗传性疾病。 其具体方法步骤在文献中 巳有多种报道， 如可参阅文献 DeRi s i, J. L. , Lyer, V. &Brown, P. 0.
(1997) Science278, 680-686.及文献 Hel le, R. A. , Schema, M. , Chai, A.， Sha lom, D. , (1997) PNAS 94: 2150-2155.
(一 ) 点样
各种不同的全长 cDNA共计 4000条多核苷酸序列作为靶 DNA,其中包括本发明 的多核苷酸。 将它们分别通过 PCR 进行扩增， 纯化所得扩增产物后将其浓度调到 500ng/ul左右， 用 Cartes ian 7500点样仪（购自美国 Cartes ian公司）点于玻璃介 质上， 点与点之间的距离为 280 μ ηι。 将点样后的玻片进行水合、 干燥、 置于紫外 交联仪中交联， 洗脱后干燥使 DNA固定在玻璃片上制备成芯片。其具体方法步骤在 文献中已有多种报道， 本实施例的点样后处理步骤是：
1. 潮湿环境中水合 4小时；
2. 0. 2%SDS洗涤 1分钟；
3. dd 0洗涤两次， 每次 1分钟；
4. NaBH4封闭 5分钟；
5. 95°C水中 2分钟；
6. 0. 2%SDS洗涤 1分钟；
7. ddH20冲洗两次；
8. 凉干， 25°C储存于暗处备用。
(二）探针标记
用一步法分别从人体混合组织与机体特定组织 （或经过刺激的细胞株） 中抽 提总 mRNA, 并用 Ol igotex mRNA Midi Ki t (购自 QiaGen公司）纯化 mRNA,通过反转 录分另 1|将焚光试亦 J Cy3dUTP (5-Araino-propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy3 f luorescent dye, 购自 Amersham Phamacia Biotech公司)标记 人体混合组织的 mRNA,用荧光试剂 Cy5dUTP (5- Amino- propargy 1-2'- deoxyur idine 5'-tr iphate coupled to Cy5 f luorescent dye, 购自 Amersham Phamacia Biotech 公司）标记机体特定组织 （或经过刺激的细胞株） mRNA, 经纯化后制备出探针。 具 体步骤参照及方法见： Schena,
M. , Shalon, D. , Hel ler, R. (1996) Proc. Nat l. Acad. Sci. USA. Vol. 93: 10614-10619. S chena, M. , Shalon, Dari. , Davi s, R. W. (1995) Science. 270. (20) : 467-480. (三） 杂交
分别将来自 以上两种组织的探针与芯片一起在 UniHyb™ Hybr idizat ion Solut ion (购自 TeleChem公司）杂交液中进行杂交 16 小时， 室温用洗涤液 （l x SSC, 0. 2%SDS ) 洗涤后用 ScanArray 3000扫描仪（购自美国 General Scanning公 司）进行扫描， 扫描的图象用 Imagene软件（美国 Biodi scovery公司）进行数据 分析处理， 算出每个点的 Cy3/Cy5比值。
以上机体特定组织（或经过刺激的细胞株）分别为膀胱粘膜、 PMA+的 ECV304细 胞株、 LPS+的 Ecv304细胞株胸腺、 正常成纤维细胞 1024NC、 Fibroblas t , 生长因子 刺激， 1024NT, 疤痕成 fc生长因子刺激， 1013HT, 疤痕成 fc未用生长因子刺激， 1013HC, 膀胱癌建株细胞 EJ、 膀胱癌旁、 膀胱癌、 肝癌、 肝癌细胞株、 胎皮、 脾脏、 前列腺癌、 空肠腺癌、 贲门癌。 根据这 17个 Cy3/Cy5比值绘出折方图。 （图 1 ) 。 由 图可见本发明所述的人 tre致癌基因蛋白 10. 56和人 tre致癌基因蛋白表达谱很相
工业实用性
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗恶性肿瘤、 肾上腺缺乏症、 皮肤病、 各类炎症、 HIV感染和免疫性 疾病等。
研究显示， 在典型的骨骼及非同寻常的骨骼外尤文氏肉瘤中显示了一种连贯 的染色体易位 t (l l; 22) (q24; (il2) , 但在小圆细胞组织的其他肿瘤中未观察到 这一现象。 并且迄今为止， t ( l l ; 22 )被认为是诊断尤文氏肉瘤唯一阳性且可 靠的标准 （Sandberg et a l. , 1988 ) 。 临床观察显示染色体易位导致异常地转 录激活基因从而启动或引发恶性状态。 这一重要的具有致瘤效应的基因被称为 tre致癌基因， 在人体尤文氏肉瘤细胞中发现， 并可在 NIH3T3细胞中产生， 但 在人体其他细胞中未曾发现。 t re致癌基因是一种可从转染人尤文氏肉瘤 DNA的 NIH3T3细胞中分离得到的重组基因。 Tre- 2是 tre致癌基因的一种新的转录单 位， 该转录单位相同的或多态的或肿瘤特异的的模型存在于人体的各种癌细胞 中， 但在正常的组织衍生细胞中没有表达。
本发明的多肽的表达谱与人 tre致癌基因蛋白的表达谱相一致， 两者具有相 似的生物学功能。 本发明的多肽在体内参与调控或启动人体多种肿瘤基因， 其 基因序列作为一种转录单位它的易位对于尤文氏肉瘤具有非常明显的激活效 应。 其表达异常通常与多种肿瘤的发生、 炎症异常或免疫调节紊乱密切相关， 并产生相关的疾病尤其是尤文氏肉瘤。
由此可见， 本发明的人 t re致癌基因蛋白 10. 56的表达异常将产生各种疾病 尤其是骨骼肿瘤、 其它肿瘤、 炎症、 免疫性疾病， 这些疾病包括但不限于：
骨骼肿瘤： 尤文氏肉瘤、 骨样骨瘤、 骨软骨瘤、 软骨瘤、 骨母细胞瘤、 软骨 母细胞瘤等） ， 恶性骨肿瘤如骨巨细胞瘤、 骨肉瘤、 软骨肉瘤、 骨髓瘤
其它肿瘤： 胃癌， 肝癌， 肺癌， 食管癌， 乳腺癌， 白血病， 淋巴瘤， 甲状腺 肿瘤， 子宫肌瘤， 星形细胞瘤， 室管膜瘤， 胶质细胞瘤， 神经纤维瘤， 结肠癌， 膀胱癌， 子宫内膜癌， 结肠癌， 喉癌， 气管肿瘤， 纤维瘤， 纤维肉瘤
炎症： 慢性活动性肝炎， 结节病， 多肌炎， 慢性鼻炎， 慢性胃炎， 脑脊髓多 发性硬化， 肾小球性肾炎， 心肌炎， 心肌病， 动脉粥样硬化， 胃溃疡， 子宫颈 炎， 各种感染性炎症
免疫性疾病： 系统性红斑狼疮， 类风湿性关节炎， 支气管哮喘， 荨麻疹， 特 异性皮炎， 感染后心肌炎， 硬皮病， 重症肌无力， 格林-巴利综合症， 普通易变 免疫缺陷病， 原发性 B淋巴细胞免疫缺陷病， 获得性免疫缺陷综合症
本发明的人 t re致癌基因蛋白 10. 56的表达异常还将产生某些遗传性，血液性 疾病等。
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗各种疾病尤其是骨骼肿瘤、 其它肿瘤、 炎症、 免疫性疾病， 某些 遗传性， 血液性疾病等。
本发明也提供了筛选化合物以鉴定提高（激动剂）或阻遏（拮抗剂）人 t re 致 癌基因蛋白 10. 56的药剂的方法。激动剂提高人 t re致癌基因蛋白 10. 56刺激细 胞增殖等生物功能， 而拮抗剂阻止和治疗与细胞过度增殖有关的紊乱如各种癌 症。例如，能在药物的存在下，将哺乳动物细胞或表达人 tre致癌基因蛋白 10. 56 的膜制剂与标记的人 tre致癌基因蛋白 1 0. 56一起培养。然后测定药物提高或阻 遏此相互作用的能力。
人 t re致癌基因蛋白 10. 56的拮抗剂包括筛选出的抗体、化合物、 受体缺失 物和类似物等。人 t re致癌基因蛋白 10. 56的拮抗剂可以与人 t re致癌基因蛋白 10. 56结合并消除其功能， 或是抑制该多肽的产生， 或是与该多肽的活性位点结 合使该多肽不能发挥生物学功能。 .
在筛选作为拮抗剂的化合物时，可以将人 tre致癌基因蛋白 10. 56加入生物 分析测定中，通过测定化合物对人 tre致癌基因蛋白 10. 56和其受体之间相互作 用的影响来确定化合物是否是拮抗剂。 用上述筛选化合物的同样方法， 可以筛选 出起拮抗剂作用的受体缺失物和类似物。能与人 tre致癌基因蛋白 10.56结合的 多肽分子可通过筛选由各种可能组合的氨基酸结合于固相物组成的随机多肽库 而获得。 筛选时， 一般应对人 tre致癌基因蛋白 10.56分子进行标记。
本发明提供了用多肽， 及其片段、 衍生物、 类似物或它们的细胞作为抗原以 生产抗体的方法。 这些抗体可以是多克隆抗体或单克隆抗体。 本发明还提供了针 对人 tre致癌基因蛋白 10.56抗原决定簇的抗体。 这些抗体包括（但不限于）： 多 克隆抗体、 单克隆抗体、 嵌合抗体、 单链抗体、 Fab片段和 Fab表达文库产生的 片段。
多克隆抗体的生产可用人 tre致癌基因蛋白 10.56直接注射免疫动物（如家 兔， 小鼠， 大鼠等） 的方法得到， 多种佐剂可用于增强免疫反应， 包括但不限于 弗氏佐剂等。制备人 tre致癌基因蛋白 10.56的单克隆抗体的技术包括但不限于 杂交瘤技术（Kohler and Milstein. Nature, 1975, 256: 495-497) , 三瘤技术， 人 Β-细胞杂交瘤技术， EBV-杂交瘤技术等。 将人恒定区和非人源的可变区结合 的嵌合抗体可用已有的技术生产（Morrison et al ， PNAS, 1985, 81: 6851)。 而已 有的生产单链抗体的技术（U.S. Pat No.4946778)也可用于生产抗人 tre致癌基 因蛋白 10.56的单链抗体。
抗人 tre致癌基因蛋白 10.56的抗体可用于免疫组织化学技术中，检测活检 标本中的人 tre致癌基因蛋白 10.56。
与人 tre致癌基因蛋白 10.56结合的单克隆抗体也可用放射性同位素标记， 注入体内可跟踪其位置和分布。这种放射性标记的抗体可作为一种非创伤性诊断 方法用于肿瘤细胞的定位和判断是否有转移。
抗体还可用于设计针对体内某一特殊部位的免疫毒素。如人 tre致癌基因蛋 白 10.56高亲和性的单克隆抗体可与细菌或植物毒素（如白喉毒素， 蓖麻蛋白， 红豆碱等）共价结合。一种通常的方法是用巯基交联剂如 SPDP,攻击抗体的氨基， 通过二硫键的交换， 将毒素结合于抗体上， 这种杂交抗体可用于杀灭人 tre致癌 基因蛋白 10， 56阳性的细胞。
本发明中的抗体可用于治疗或预防与人 tre 致癌基因蛋白 10.56相关的疾 病。给予适当剂量的抗体可以刺激或阻断人 tre致癌基因蛋白 10.56的产生或活 性。
本发明还涉及定量和定位检测人 tre致癌基因蛋白 10.56水平的诊断试验方 法。 这些试验是本领域所熟知的， 且包括 FISH测定和放射免疫测定。 试验中所 检测的人 tre致癌基因蛋白 10.56水平，可以用作解释人 tre致癌基因蛋白 10.56 在各种疾病中的重要性和用于诊断人 tre致癌基因蛋白 10.56起作用的疾病。 本发明的多肽还可用作肽谱分析， 例如， 多肽可用物理的、 化学或酶进行特 异性切割， 并进行一维或二维或三维的凝胶电泳分析，更好的是进行质谱分析。
编码人 tre致癌基因蛋白 10.56的多核苷酸也可用于多种治疗目的。基因治 疗技术可用于治疗由于人 tre致癌基因蛋白 10.56的无表达或异常 /无活性表达 所致的细胞增殖、 发育或代谢异常。 重组的基因治疗载体 (如病毒载体）可设计用 于表达变异的人 tre致癌基因蛋白 10.56, 以抑制内源性的人 tre致癌基因蛋白 10.56活性。 例如， 一种变异的人 tre致癌基因蛋白 10.56可以是缩短的、 缺失 了信号传导功能域的人 tre致癌基因蛋白 10.56, 虽可与下游的底物结合， 但缺 乏信号传导活性。 因此重组的基因治疗载体可用于治疗人 tre 致癌基因蛋白 10.56表达或活性异常所致的疾病。 来源于病毒的表达载体如逆转录病毒、 腺病 毒、 腺病毒相关病毒、 单纯疱疹病毒、 细小病毒等可用于将编码人 tre致癌基因 蛋白 10.56的多核苷酸转移至细胞内。 构建携带编码人 tre致癌基因蛋白 10.56 的多核苷酸的重组病毒载体的方法可见于已有文献（Sambrook,et al.)0 另外重 组编码人 tre致癌基因蛋白 10.56的多核苷酸可包装到脂质体中转移至细胞内。
多核苷酸导入组织或细胞内的方法包括： 将多核苷酸直接注入到体内组织 中； 或在体外通过载体（如病毒、 噬菌体或质粒等）先将多核苷酸导入细胞中， 再 将细胞移植到体内等。
抑制人 tre致癌基因蛋白 10.56 mRNA的寡核苷酸（包括反义 RNA和 DNA)以 及核酶也在本发明的范围之内。核酶是一种能特异性分解特定 RNA的酶样 RNA分 子， 其作用机制是核酶分子与互补的靶 RNA特异性杂交后进行核酸内切作用。 反 义的 RNA和 DNA及核酶可用已有的任何 RNA或 DM合成技术获得，如固相磷酸酰 胺化学合成法合成寡核苷酸的技术已广泛应用。 反义 RNA分子可通过编码该 RNA 的 DNA序列在体外或体内转录获得。这种 DNA序列已整合到载体的 RNA聚合酶启 动子的下游。 为了增加核酸分子的稳定性， 可用多种方法对其进行修饰， 如增加 两侧的序列长度， 核糖核苷之间的连接应用磷酸硫酯键或肽键而非磷酸二酯键。
编码人 tre致癌基因蛋白 10.56 的多核苷酸可用于与人 tre致癌基因蛋白 10.56的相关疾病的诊断。 编码人 tre致癌基因蛋白 10.56的多核苷酸可用于检 测人 tre 致癌基因蛋白 10.56 的表达与否或在疾病状态下人 tre致癌基因蛋白 10.56的异常表达。 如编码人 tre致癌基因蛋白 10.56的 DNA序列可用于对活检 标本进行杂交以判断人 tre 致癌基因蛋白 10.56 的表达状况。 杂交技术包括 Southern 印迹法， Northern印迹法、 原位杂交等。 这些技术方法都是公开的成 熟技术， 相关的试剂盒都可从商业途径得到。 本发明的多核苷酸的一部分或全部 可作为探针固定在微阵列（Microarray)或 DNA芯片（又称为 "基因芯片"）上， 用 于分析组织中基因的差异表达分析和基因诊断。用人 tre致癌基因蛋白 10. 56特 异的引物进行 MA-聚合酶链反应（RT- PCR)体外扩增也可检测人 tre 致癌基因蛋 白 10. 56的转录产物。
检测人 t re致癌基因蛋白 10. 56基因的突变也可用于诊断人 tre致癌基因蛋 白 10. 56相关的疾病。人 tre致癌基因蛋白 10. 56突变的形式包括与正常野生型 人 tre致癌基因蛋白 10. 56 DM序列相比的点突变、 易位、 缺失、 重组和其它任 何异常等。 可用已有的技术如 Southern印迹法、 DNA序列分析、 PCR和原位杂交 检测突变。另外，突变有可能影响蛋白的表达， 因此用 Northern印迹法、 Wes tern 印迹法可间接判断基因有无突变。
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人 染色体具体位置且并可以与其杂交。 目前， 需要鉴定染色体上的各基因的具体 位点。 现在， 只有很少的基于实际序列数据（重复多态性）的染色体标记物可用 于标记染色体位置。 根据本发明， 为了将这些序列与疾病相关基因相关联， 其 重要的第一步就是将这些 DNA序列定位于染色体上。
简而言之， 根据 cDM制备 PCR引物（优选 15- 35bp)， 可以将序列定位于染色 体上。 然后， 将这些引物用于 PCR筛选含各条人染色体的体细胞杂合细胞。 只有 那些含有相应于引物的人基因的杂合细胞会产生扩增的片段。
体细胞杂合细胞的 PCR定位法， 是将 DNA定位到具体染色体的快捷方法。 使 用本发明的寡核苷酸引物， 通过类似方法， 可利用一组来自特定染色体的片段 或大量基因组克隆而实现亚定位。 可用于染色体定位的其它类似策略包括原位 杂交、 用标记的流式分选的染色体预筛选和杂交预选， 从而构建染色体特异的 cDNA库。
将 cDNA克隆与中期染色体进行荧光原位杂交（FISH) , 可以在一个步骤中精 确地进行染色体定位。 此技术的综述， 参见 Verma等， Human Chromosomes: a Manua l of Bas ic Techniques, Pergamon Pres s, New York (1988)。
一旦序列被定位到准确的染色体位置， 此序列在染色体上的物理位置就可 以与基因图数据相关联。 这些数据可见于例如， V. Mckus ick, Mende l ian Inher i tance in Man (可通过与 Johns Hopkins Univers i ty Wel ch Medi ca l Library联机获得）。 然后可通过连锁分析， 确定基因与业已定位到染色体区域 上的疾病之间的关系。 接着， 需要测定患病和未患病个体间的 cDNA或基因组序列差异。 如果在一 些或所有的患病个体中观察到某突变， 而该突变在任何正常个体中未观察到， 则该突变可能是疾病的病因。 比较患病和未患病个体， 通常涉及首先寻找染色 体中结构的变化，如从染色体水平可见的或用基于 cDM序列的 PCR可检测的缺失 或易位。 根据目前的物理作图和基因定位技术的分辨能力， 被精确定位至与疾 病有关的染色体区域的 cDNA , 可以是 50至 500个潜在致病基因间之一种（假定 1 兆碱基作图分辨能力和每 20kb对应于一个基因）。
可以将本发明的多肽、 多核苷酸及其模拟物、 激动剂、 拮抗剂和抑制剂与合 适的药物载体组合后使用。 这些载体可以是水、 葡萄糖、 乙醇、 盐类、 缓冲液、 甘油以及它们的组合。组合物包含安全有效量的多肽或拮抗剂以及不影响药物效 果的载体和赋形剂。 这些组合物可以作为药物用于疾病治疗。
本发明还提供含有一种或多种容器的药盒或试剂盒，容器中装有一种或多种 本发明的药用组合物成分。 与这些容器一起， 可以有由制造、 使用或销售药品或 生物制品的政府管理机构所给出的指示性提示， 该提示反映出生产、 使用或销售 的政府管理机构许可其在人体上施用。 此外， 本发明的多肽可以与其它的治疗化 合物结合使用。
药物组合物可以以方便的方式给药， 如通过局部、 静脉内、 腹膜内、 肌内、 皮下、 鼻内或皮内的给药途径。 人 t re致癌基因蛋白 10. 56 以有效地治疗和 /或 预防具体的适应症的量来给药。施用于患者的人 t re致癌基因蛋白 1 0. 56的量和 剂量范围将取决于许多因素， 如给药方式、待治疗者的健康条件和诊断医生的判

Claims

杈 利 要 求 书
1、 一种分离的多肽-人 tre致癌基因蛋白 10. 56 , 其特征在于它包含有： SEQ ID NO: 2所示的氨基酸序列的多肽、 或其多肽的活性片段、 类似物或衍生物。
2、 如权利要求 1所述的多肽， 其特征在于所述多肽、 类似物或衍生物的氨基酸 序列具有与 SEQ ID NO: 2所示的氨基酸序列至少 95°/。的相同性。
3、 如权利要求 2所述的多肽， 其特征在于它包含具有 SEQ ID NO: 2所示的氨基 酸序列的多肽。
4、 一种分离的多核苷酸， 其特征在于所述多核苷酸包含选自下组中的一种：
(a) 编码具有 SEQ ID NO: 2所示氨基酸序列的多肽或其片段、 类似物、 衍生 物的多核苷酸；
(b) 与多核苷酸 （a ) 互补的多核苷酸； 或
(c) 与 （a ) 或 （b ) 有至少 70%相同性的多核苷酸。
5、 杈利要求 4所述的多核苷酸， 其特征在于所述多核苷酸包含编码具有 SEQ ID NO: 2所示氨基酸序列的多核苷酸。
6、 如权利要求 4所述的多核苷酸， 其特征在于所述多核苷酸的序列包含有 SEQ ID NO: 1 中 904-1194位的序列或 SEQ ID NO: 1中 1-1421位的序列。
7、 一种含有外源多核苷酸的重组载体， 其特征在于它是由权利要求 4-6中的任 一权利要求所述多核苷酸与质粒、 病毒或运载体表达载体构建而成的重组载体。
8、 一种含有外源多核苷酸的遗传工程化宿主细胞， 其特征在于它是选自于下列 一种宿主细胞：
(a) 用权利要求 7所述的重组载体转化或转导的宿主细胞； 或
(b) 用权利要求 4-6 中的任一权利要求所述多核苷酸转化或转导的宿主细 胞。
9、 一种具有人 tre致癌基因蛋白 10. 56活性的多肽的制备方法， 其特征在于所 述方法包括：
(a) 在表达人 t re致癌基因蛋白 1 0. 56条件下， 培养权利要求 8所述的工程 化宿主细胞；
(b) 从培养物中分离出具有人 t re致癌基因蛋白 10. 56活性的多肽。
1 0、 一种能与多肽结合的抗体，其特征在于所述抗体是能与人 t re致癌基因蛋白 10. 56特异性结合的抗体。
11、 一类模拟或调节多肽活性或表达的化合物， 其特征在于它们是模拟、 促进、 拮抗或抑制人 t re致癌基因蛋白 10. 56的活性的化合物。
12、 如权利要求 11所述的化合物， 其特征在于它是 SEQ ID NO: 1所示的多核苷 酸序列或其片段的反义序列。
1 3、一种权利要求 11所述化合物的应用，其特征在于所述化合物用于调节人 t re 致癌基因蛋白 10. 56在体内、 体外活性的方法。
14、 一种检测与权利要求 1-3 中的任一权利要求所述多肽相关的疾病或疾病易 感性的方法， 其特征在于其包括检测所述多肽的表达量， 或者检测所述多肽的 活性， 或者检测多核苷酸中引起所述多肽表达量或活性异常的核苷酸变异。
15、 如权利要求 1-3 中的任一权利要求所述多肽的应用， 其特征在于它应用于 筛选人 t re致癌基因蛋白 10. 56 的模拟物、 激动剂， 拮抗剂或抑制剂； 或者用 于肽指紋图谱鉴定。
16、 如杈利要求 4-6 中的任一杈利要求所述的核酸分子的应用， 其特征在于它 作为引物用于核酸扩增反应， 或者作为探针用于杂交反应， 或者用于制造基因 芯片或微阵列。
17、 如杈利要求 1-6及 11中的任一权利要求所述的多肽、 多核苷酸或化合物的 应用， 其特征在于用所述多肽、 多核苷酸或其模拟物、 激动剂、 拮抗剂或抑制 剂以安全有效剂量与药学上可接受的载体组成作为诊断或治疗与人 tre 致癌基 因蛋白 1 0. 56异常相关的疾病的药物组合物。
18、 权利要求 1-6及 11中的任一权利要求所述的多肽、 多核苷酸或化合物的应 用， 其特征在于用所述多肽、 多核苷酸或化合物制备用于治疗如恶性肿瘤， 血 液病， HIV感染和免疫性疾病和各类炎症的药物。
PCT/CN2001/000936 2000-06-12 2001-06-11 Nouveau polypeptide, proteine humaine 10.56 du gene cancerigene tre, et polynucleotide codant ce polypeptide WO2002000824A2 (fr)

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CN 00116436 CN1328029A (zh) 2000-06-12 2000-06-12 一种新的多肽——人tre致癌基因蛋白10.56和编码这种多肽的多核苷酸
CN00116436.8 2000-06-12

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3936620A1 (en) 2012-04-06 2022-01-12 Geneohm Sciences Canada, Inc. Sequences for detection and identification of methicillin-resistant staphylococcus aureus (mrsa) of mrej type xxi

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] 21 December 1999 ABU-THREIDEH, J. ET AL. Retrieved from NCBI, accession no. GI: 3980546 Database accession no. (AC004875.1) *
DATABASE GENBANK [Online] 22 February 2000 ISOGAI, T. ET AL. Retrieved from NCBI, accession no. GI: 7023820 Database accession no. (BAA92096.1) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3936620A1 (en) 2012-04-06 2022-01-12 Geneohm Sciences Canada, Inc. Sequences for detection and identification of methicillin-resistant staphylococcus aureus (mrsa) of mrej type xxi

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